Use of plekhh2 in the diagnosis and treatment of pulmonary arterial hypertension

Abstract

The use of PLEKHH2 in the diagnosis and treatment of pulmonary arterial hypertension. It is found for the first time that PLEKHH2 is a novel pulmonary arterial hypertension genetic related gene, and upregulation of the expression of the PLEKHH2 gene can effectively prevent and reverse pulmonary arterial hypertension, thus providing a novel treatment target for the treatment of pulmonary arterial hypertension, and achieving significant clinical application value. In addition, it is found for the first time that inhibiting the binding of Hic-5 to SMAD7 or inhibiting Hic-5 is effective for the treatment of pulmonary arterial hypertension, thus providing a novel thought and policy for screening and research and development of drugs treating pulmonary arterial hypertension.

Description

Application of PLEKHH2 in the diagnosis and treatment of pulmonary hypertension

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the following application documents: invention patent application number 202310978261X, filed on August 4, 2023, entitled “Application of PLEKHH2 in the Diagnosis and Treatment of Pulmonary Arterial Hypertension,” the contents of which are incorporated by reference in their entirety. Technical Field

[0003] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of PLEKHH2 in the diagnosis and treatment of pulmonary hypertension. Background Art

[0004] Pulmonary arterial hypertension (PAH) is a malignant pulmonary vascular disease characterized by elevated pulmonary artery pressure and progressively increased pulmonary vascular resistance, often leading to severe right heart failure and even death. Idiopathic pulmonary arterial hypertension (IPAH) and hereditary pulmonary arterial hypertension (HPAH) are the most common types of PAH, accounting for 37%-49% of the first major type of PAH. IPAH / HPAH are rare diseases, with an incidence of 5-10 cases per million people. IPAH / HPAH can occur in all age groups, from children to adults. The primary clinical challenge facing IPAH / HPAH is the lack of truly effective treatments. Existing pulmonary hypertension treatments are symptomatic treatments that dilate pulmonary vasculature and can only slow disease progression; no drug can effectively reverse the pathological pulmonary vascular remodeling. The significant disease burden of IPAH / HPAH has attracted high attention at the national level. In 2018, the National Health Commission and five other ministries included IPAH / HPAH in the "First List of Rare Diseases in China", clearly defining it as a national important rare disease. Therefore, it is imperative to further accelerate basic research related to pulmonary arterial hypertension and discover new drug targets.

[0005] IPAH / HPAH is a monogenic autosomal dominant genetic disease with obvious familial inheritance and aggregation. As of December 2022, a total of 21 pulmonary hypertension genetic-related genes have been discovered worldwide (of which BMP9 and PTGIS were originally reported by the research group of the inventor of the present invention), which can generally explain the causes of 50-70% of HPAH and 20-40% of IPAH patients. Genetically related genes are not only a breakthrough in understanding the cause of pulmonary hypertension patients, but also the key "key" to uncovering the pathological mechanism of pulmonary hypertension and developing new drugs. Most of the currently known pathogenic genes for pulmonary hypertension (BMPR2, ALK1, ENG, CAV1, SOX17, KDR, etc.) are highly expressed in pulmonary vascular endothelial cells, suggesting that pulmonary vascular endothelial cell damage is the source of pulmonary hypertension. Among them, targeting the pathogenic genes BMPR2 and SOX17 of pulmonary arterial hypertension, upregulating the expression of these genes or increasing their activity can protect pulmonary arterial endothelial cells, inhibit proliferation, and reverse mesenchymal transition. It has shown good effects in preventing or treating pulmonary arterial hypertension in animal models. Some drugs have entered clinical trials and are currently a hot spot in the global research and development of pulmonary arterial hypertension drugs.

[0006] Therefore, the discovery of genetically associated genes for pulmonary hypertension has dual clinical significance: first, pathogenic mutations are the true cause of IPAH / HPAH pulmonary hypertension and are crucial for clinical diagnosis; second, if it is confirmed that the pathogenic gene has a protective effect, it will become a new potential target, providing new possibilities for clinical treatment. In view of this, the present invention, using a "genetic family discovery-sporadic patient validation" strategy, first discovered PLEKHH2 as a novel genetically associated gene for pulmonary hypertension. Cell experiments demonstrated that the PLEKHH2 gene has an important protective effect on pulmonary vascular endothelial cells, and animal experiments confirmed that high expression of PLEKHH2 can effectively prevent and treat pulmonary hypertension. To date, there have been no studies or reports on the use of PLEKHH2 in the diagnosis and / or treatment of pulmonary arterial hypertension.

[0007] Summary of the Invention

[0008] In view of the technical problems existing in the prior art, the purpose of the present invention is to provide an application of PLEKHH2 in the diagnosis and treatment of pulmonary hypertension.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A first aspect of the present invention provides an agent that inhibits the binding of Hic-5 to SMAD7 and / or the use of an agent that inhibits Hic-5 in the preparation of a medicament for preventing, treating, alleviating and / or improving pulmonary hypertension.

[0011] Furthermore, the reagents include small molecule compounds, polypeptides, peptide mimetics, nucleic acid molecules, proteins, antibodies, antibody mimetics, fusion proteins, protein analogs, gene delivery vectors, protein delivery vectors, aptamers, shRNA, siRNA, miRNA, antisense nucleic acids, CRISPR gene editing reagents and / or any combination thereof targeting the Hic-5 and SMAD7 binding sites and / or targeting Hic-5;

[0012] Optionally, the Hic-5 and SMAD7 binding site is the LIM3 domain of Hic-5; optionally, the reagent can compete with SMAD7 to bind to the LIM3 domain of Hic-5; optionally, the reagent includes PLEKHH2 and / or a PLEKHH2 promoter; optionally, the PLEKHH2 and / or a PLEKHH2 promoter includes a PLEKHH2 gene, a PLEKHH2 mRNA, a PLEKHH2 cDNA, PLEKHH2 protein, peptide fragments of PLEKHH2 protein, analogs of PLEKHH2 protein, active fragments of any of the foregoing, vectors expressing PLEKHH2, nanoparticles carrying PLEKHH2 gene, viral vectors carrying PLEKHH2 gene, PEG-modified proteins encapsulating PLEKHH2 gene or protein, protein microspheres encapsulating PLEKHH2 gene or protein, liposomes encapsulating PLEKHH2 gene or protein, extracellular vesicles encapsulating PLEKHH2 gene or protein and / or any combination thereof; optionally, the PLEKHH2 and / or PLEKHH2 promoter prevents, treats, alleviates and / or improves pulmonary arterial hypertension by inhibiting SMAD2 / 3 hyperphosphorylation.

[0013] In the present invention, the present invention experimentally verifies for the first time the effectiveness of inhibiting the binding of Hic-5 to SMAD7 and / or inhibiting Hic-5 in preventing, treating, alleviating and / or improving pulmonary hypertension.

[0014] A second aspect of the present invention provides an agent that inhibits the binding of Hic-5 to SMAD7 and / or the use of an agent that inhibits Hic-5 in preventing, treating, alleviating and / or improving pulmonary hypertension.

[0015] Furthermore, the reagent is a reagent that inhibits the binding of Hic-5 to SMAD7 and / or a reagent that inhibits Hic-5 as described above.

[0016] The third aspect of the present invention provides the use of PLEKHH2 and / or a PLEKHH2 promoter in the preparation of a medicament for preventing, treating, alleviating and / or improving pulmonary hypertension.

[0017] Further, the PLEKHH2 includes PLEKHH2 gene, PLEKHH2 mRNA, PLEKHH2 cDNA, PLEKHH2 protein, active fragments of any of the foregoing, and / or any combination thereof; optionally, the PLEKHH2 promoter includes a substance that increases the level of PLEKHH2, a substance that enhances the activity of PLEKHH2, a substance that delays the metabolism of PLEKHH2, and / or any combination thereof; optionally, the PLEKHH2 promoter includes a natural purified substance, a modified natural purified substance, a semi-synthetic substance, and / or a chemically synthesized substance; optionally, the PLEKHH2 promoter includes PLEKHH2 protein, a peptide segment of PLEKHH2 protein, an analog of PLEKHH2 protein, a vector expressing PLEKHH2, a nanoparticle carrying the PLEKHH2 gene, a viral vector carrying the PLEKHH2 gene, a PEG-modified protein encapsulating the PLEKHH2 gene or protein, a protein microsphere encapsulating the PLEKHH2 gene or protein, a liposome encapsulating the PLEKHH2 gene or protein, an extracellular vesicle encapsulating the PLEKHH2 gene or protein, and / or any combination thereof. Optionally, the PLEKHH2 and / or PLEKHH2 promoter can reduce right ventricular systolic pressure, alleviate right ventricular hypertrophy, increase tricuspid annular systolic displacement, and alleviate pulmonary artery vascular remodeling.

[0018] In some embodiments, the PLEKHH2 comprises a PLEKHH2 gene and a PLEKHH2 protein. The PLEKHH2 gene is transcribed and translated into a PLEKHH2 protein product in the subject. In a preferred embodiment, the PLEKHH2 gene has a Gene ID of 130271, which is pleckstrin homology, MyTH4 and FERM domain containing H2 [Homo sapiens (human)]. The Hic-5 is also known as TGFB1I1, and its corresponding Gene ID in NCBI (https: / / www.ncbi.nlm.nih.gov / ) is 7041. The SMAD7 has a Gene ID of 4092 in NCBI.

[0019] In some embodiments, the PLEKHH2 is from a mammal, including but not limited to humans, non-human primates (e.g., gorillas, apes, monkeys), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), livestock (e.g., horses, cows, sheep, pigs, rabbits). In a preferred embodiment, the PLEKHH2 is from a human.

[0020] In some embodiments, the PLEKHH2 has a sequence known in the art or is a derivative thereof. In some embodiments, the PLEKHH2 is a molecule comprising the following sequences: (a) a PLEKHH2 molecule having a sequence such as Gene ID: 130271 (human), Gene ID: 213556 (mouse), Gene ID: 313866 (Norway rat), Gene ID: 713488 (rhesus monkey), Gene ID: 100299044 (cattle); (b) a molecule that hybridizes to the sequence defined in (a) under stringent conditions; (c) a molecule having a sequence homology of 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or more, or any value or range of values ​​therebetween) with the sequence of the molecule shown in (a) or (b), such as a PLEKHH2 molecule obtained by codon optimization.

[0021] In some embodiments, the PLEKHH2 promoter refers to a substance that can increase the level of PLEKHH2, enhance the activity of PLEKHH2 and / or delay the metabolism of PLEKHH2, including but not limited to: small molecule compounds, vectors expressing PLEKHH2, nanoparticles carrying the PLEKHH2 gene, viral vectors carrying the PLEKHH2 gene, PEG-modified proteins encapsulating the PLEKHH2 gene or protein, protein microspheres encapsulating the PLEKHH2 gene or protein, liposomes encapsulating the PLEKHH2 gene or protein, extracellular vesicles encapsulating the PLEKHH2 gene or protein and / or any combination thereof.

[0022] In some embodiments, the vector includes but is not limited to: a lentiviral vector, a retroviral vector, a poxvirus vector, a herpes simplex virus vector, an adenovirus vector, an adeno-associated virus vector, a DNA plasmid vector, a liposome bound to a DNA plasmid, a molecular couple bound to a DNA plasmid and / or a polymer bound to a DNA plasmid. As long as the vector can be used to deliver the target gene PLEKHH2 described in the present invention, it is within the scope of protection of the present invention. In a preferred embodiment, the vector is an adenovirus vector.

[0023] The fourth aspect of the present invention provides the use of PLEKHH2 and / or a PLEKHH2 promoter in preventing, treating, alleviating and / or improving pulmonary hypertension.

[0024] Furthermore, the PLEKHH2 and / or PLEKHH2 promoter is the PLEKHH2 and / or PLEKHH2 promoter described above.

[0025] A fifth aspect of the present invention provides a pharmaceutical composition for preventing, treating, alleviating and / or improving pulmonary arterial hypertension, the pharmaceutical composition comprising an agent that inhibits the binding of Hic-5 to SMAD7 and / or an agent that inhibits Hic-5 as described above, and / or PLEKHH2 and / or a PLEKHH2 promoter as described above.

[0026] Furthermore, the pharmaceutical composition may also contain other drugs for preventing, treating, alleviating and / or improving pulmonary hypertension; optionally, the drugs include calcium channel blockers, prostacyclins, endothelin receptor antagonists, phosphodiesterase-5 inhibitors and / or guanylate cyclase agonists; optionally, the pharmaceutical composition may also contain pharmaceutically acceptable carriers and / or excipients. In some embodiments, the other drugs for preventing, treating, alleviating and / or improving pulmonary hypertension contained in the pharmaceutical composition include but are not limited to calcium channel blockers, prostacyclins, endothelin receptor antagonists, phosphodiesterase-5 inhibitors and / or guanylate cyclase agonists. As long as the drugs can be used for preventing, treating, alleviating and / or improving pulmonary hypertension, they are within the scope of protection of the present invention. In some embodiments, the calcium channel blockers include but are not limited to diltiazem, verapamil, nifedipine, amlodipine, nitrendipine, felodipine, and lercanidipine; the prostacyclin drugs include but are not limited to rioprost, abaprost, dinoprostone, cyprostatin, enprost, trimoprost, and roxaprostol; the endothelin receptor antagonists include but are not limited to bosentan, ambrisentan, and macitentan; the phosphodiesterase-5 inhibitors include but are not limited to tadalafil, vardenafil, and sildenafil; the guanylate cyclase agonists include but are not limited to rioxiguanidine, beraprost, vericiguat, and linaclotide. In some embodiments, specific illustrative examples of the pharmaceutically acceptable carriers and / or excipients include, but are not limited to: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc.

[0027] In some embodiments, suitable pharmaceutically acceptable carriers and / or excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used to help stabilize the formulation, improve its activity or bioavailability, or produce an acceptable taste or smell when orally administered. The formulations that can be used in such pharmaceutical compositions can be in the form of the original compound itself, or optionally in the form of a pharmaceutically acceptable salt thereof. The pharmaceutical composition thus formulated can be administered by any appropriate method known to those skilled in the art, as needed. When the pharmaceutical composition is used, a safe and effective amount of the pharmaceutical composition of the present invention is administered to a human.

[0028] A sixth aspect of the present invention provides a pharmaceutical preparation for preventing, treating, alleviating and / or improving pulmonary hypertension, the pharmaceutical preparation comprising the pharmaceutical composition as described above;

[0029] Optionally, the dosage form of the pharmaceutical preparation includes injection, lyophilized powder, solution, tablet, capsule, granule, ointment, cream, gel, suspension, oral solution, controlled release dosage form, nanocrystal preparation, microemulsion and / or solid dispersion.

[0030] In some embodiments, the appropriate dosage of the pharmaceutical composition or pharmaceutical preparation described in the present invention can be prescribed in a variety of ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, morbidity, diet, administration time, administration route, excretion rate and reaction sensitivity. A skilled physician can usually easily determine the prescription and the dosage that is effective for the desired prevention and / or treatment.

[0031] A seventh aspect of the present invention provides the use of the LIM3 domain of Hic-5 in screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary arterial hypertension.

[0032] The eighth aspect of the present invention provides the use of PLEKHH2 and / or Hic-5 in screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary arterial hypertension.

[0033] A ninth aspect of the present invention provides the use of Hic-5 and / or SMAD7 in screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary hypertension.

[0034] The tenth aspect of the present invention provides the use of SMAD7 in the preparation of a medicament for preventing, treating, alleviating and / or improving pulmonary hypertension.

[0035] An eleventh aspect of the present invention provides use of a reagent for detecting the expression level of PLEKHH2 in a sample in the preparation of a product for diagnosing and / or assisting in the diagnosis of pulmonary arterial hypertension.

[0036] Furthermore, the reagents include reagents for detecting the expression level of PLEKHH2 mRNA in a sample and reagents for detecting the expression level of PLEKHH2 protein in a sample; optionally, the reagents for detecting the expression level of PLEKHH2 mRNA in a sample include primers that specifically amplify PLEKHH2 and / or probes that specifically recognize PLEKHH2; optionally, the reagents for detecting the expression level of PLEKHH2 protein in a sample include binding agents that specifically bind to the protein encoded by PLEKHH2; optionally, the binding agents include antibodies, antibody functional fragments, agglutinating agents, receptors and / or conjugated antibodies that specifically bind to the protein encoded by PLEKHH2.

[0037] In some embodiments, the sample refers to a composition obtained or derived from a subject of interest, comprising cellular entities and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. The sample can be obtained from the subject's blood and other fluid samples of biological origin and tissue samples, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of the tissue sample can be solid tissue, such as from fresh, frozen, and / or preserved organ or tissue samples, biopsy tissue, or aspirates; blood or any blood component; body fluid; cells from any time during the individual's pregnancy or development; or plasma. The term sample includes biological samples that have been processed in any way after their acquisition, such as by reagent treatment, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes. The samples described in the present invention include, but are not limited to, blood, tissue, blood-derived cells, serum, plasma, lymph, synovial fluid, cell extracts, and combinations thereof. In preferred embodiments, the sample is selected from the subject's blood or tissue. In some embodiments, the binding agent that specifically binds to the protein encoded by PLEKHH2 includes an antibody, functional antibody fragment, agglutinating agent, receptor, conjugated antibody peptide, aptamer, and / or compound that specifically binds to the PLEKHH2 protein. In some embodiments, the reagent is used to detect the expression level of PLEKHH2 in a sample using sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology, or protein immunoassay technology. Furthermore, candidate drugs can be screened using the following methods.

[0038] A twelfth aspect of the present invention provides a method for screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary hypertension, the method comprising the following steps:

[0039] (a) treating a system expressing or containing the PLEKHH2 gene with a test substance;

[0040] (b) detecting the expression of the PLEKHH2 gene in the system;

[0041] (c) selecting a test substance that can increase the expression level of the PLEKHH2 gene as a candidate drug;

[0042] In some embodiments, any substance that may have a preventive, therapeutic, alleviating, and / or ameliorating effect on pulmonary arterial hypertension is a test substance. Furthermore, the test substance selected in step (c) is a test substance that significantly increases the expression level of PLEKHH2 compared to the expression level detected in the absence of the test substance.

[0043] The thirteenth aspect of the present invention provides a method for screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary hypertension based on the LIM3 domain of Hic-5, the method comprising the following steps: screening an agent that can bind to the LIM3 domain of Hic-5 as a candidate drug.

[0044] The fourteenth aspect of the present invention provides a method for screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary hypertension based on PLEKHH2 and / or Hic-5, comprising the following steps: simulating the binding site of Hic-5 and PLEKHH2, and designing an agent capable of binding to the binding site as a candidate drug.

[0045] The fifteenth aspect of the present invention provides a method for screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary hypertension based on Hic-5 and / or SMAD7, which comprises the following steps: simulating the binding site of Hic-5 and SMAD7, and designing an agent capable of binding to the binding site as a candidate drug.

[0046] Optionally, the system includes a cell system, a subcellular system, a solution system, a tissue system, an organ system and / or an animal system; optionally, the test substance includes a substance that increases the level of PLEKHH2, a substance that enhances the activity of PLEKHH2, a substance that delays the metabolism of PLEKHH2 and / or any combination thereof; optionally, the reagent includes a small molecule compound, polypeptide, peptide mimetic, nucleic acid molecule, protein, antibody, antibody mimetic, fusion protein, protein analog, gene delivery vector, protein delivery vector, aptamer, shRNA, siRNA, miRNA, antisense nucleic acid, CRISPR gene editing reagent and / or any combination thereof targeting the Hic-5 and SMAD7 binding site and / or Hic-5.

[0047] A sixteenth aspect of the present invention provides a product for diagnosing and / or assisting in the diagnosis of pulmonary hypertension, the product comprising the reagent as described above;

[0048] Optionally, the product further comprises a reagent for detecting the expression level of PLEKHH2 in a sample by sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology and / or protein immunoassay technology; optionally, the product comprises a test kit, a chip and / or a test strip. Further, the test kit comprises an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid detection kit or an MRM (multiple reaction monitoring) kit; in some embodiments, the test kit may further comprise elements necessary for reverse transcription polymerase chain reaction. The RT-PCR kit comprises a pair of primers specific for a gene encoding a marker protein. Each primer is a nucleotide having a nucleic acid sequence specific for the gene, and its length may be about 7 to 50 bp, more particularly about 10-39 bp. In addition, the test kit may further comprise a primer specific for the nucleic acid sequence of a control gene. In some embodiments, the RT-PCR kit may also include test tubes or suitable containers, reaction buffer (different pH values ​​and magnesium concentrations), deoxynucleotides (dNTPs), enzymes (such as Taq polymerase and reverse transcriptase), deoxyribonuclease inhibitors, ribonuclease inhibitors, DEPC-water and sterile water. In some embodiments, the kit may include the necessary elements for operating a DNA chip. The DNA chip kit may include a substrate that binds to a gene or cDNA or an oligonucleotide equivalent to a fragment thereof, and reagents, agents and enzymes for constructing a fluorescently labeled probe. In addition, the substrate may include a control gene or cDNA or an oligonucleotide equivalent to a fragment thereof. In some embodiments, the kit disclosed in the present invention may include the necessary elements for performing an ELISA. The ELISA kit may include an antibody specific for a protein (the biomarker PLEKHH2 protein described in the present invention). The antibody has high selectivity and affinity for the marker protein, no cross-reactivity with other proteins, and can be a monoclonal antibody, a polyclonal antibody or a recombinant antibody. In addition, the ELISA kit may include an antibody specific for a control protein. In addition, the ELISA kit may further comprise a reagent capable of detecting the bound antibody, for example, a labeled secondary antibody, a chromophore, an enzyme (eg, conjugated to the antibody), and a substrate thereof or a substance capable of binding to the antibody.

[0049] The seventeenth aspect of the present invention provides a system / device for diagnosing and / or assisting in the diagnosis of pulmonary arterial hypertension, the system / device comprising a processor, an input module, and an output module; wherein the processor is used to perform logical operations on the input information using bioinformatics methods; the input module is used to input the expression level of PLEKHH2 in the subject's sample, and contains a computer-readable medium of instructions, which, when executed by the processor, execute an algorithm on the input expression level of PLEKHH2; the output module is used to output whether the subject suffers from pulmonary arterial hypertension or the risk of suffering from pulmonary arterial hypertension.

[0050] An eighteenth aspect of the present invention provides a method for preventing, treating, alleviating and / or improving pulmonary arterial hypertension, comprising the steps of administering to a subject in need thereof an effective amount of an agent for inhibiting the binding of Hic-5 to SMAD7 and / or an agent for inhibiting Hic-5, PLEKHH2 and / or a PLEKHH2 promoter, a pharmaceutical composition and / or a pharmaceutical preparation as described above of the present invention.

[0051] The nineteenth aspect of the present invention provides a method for diagnosing and / or assisting in the diagnosis of pulmonary arterial hypertension, the method comprising the following steps: detecting the expression level of PLEKHH2 in a sample derived from a subject; if the expression level of PLEKHH2 in the sample derived from the subject is significantly lower than that of a normal person, the subject is diagnosed as a patient with pulmonary arterial hypertension.

[0052] In some embodiments, the subject refers to any animal, including humans and non-human animals. The term non-human animals includes all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc. In a specific embodiment of the present invention, the subject is preferably a human.

[0053] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0054] (1) The present invention adopts the strategy of "genetic family discovery-sporadic patient verification" to discover for the first time that PLEKHH2 is a new genetic gene related to pulmonary arterial hypertension. The results of gene function studies show that PLEKHH2 is specifically expressed in pulmonary arterial endothelial cells and has the potential function of regulating the activity of the BMPR2 / TGF-β pathway and protecting pulmonary arterial endothelial cells. In addition, the present invention found that the PELKHH2 gene is significantly downregulated under the pathological conditions of pulmonary arterial hypertension. In an animal model of pulmonary arterial hypertension, upregulating the expression of the PLEKHH2 gene using adenovirus can effectively prevent and reverse pulmonary arterial hypertension caused by monocrotaline (MCT), confirming the effectiveness of PLEKHH2 in preventing, treating and / or diagnosing pulmonary arterial hypertension, providing a new therapeutic target for the treatment of pulmonary arterial hypertension, and having important clinical application value.

[0055] (2) The present invention discovered for the first time that PLEKHH2 and SMAD7 competitively bind to Hic-5 protein in lung tissue. Under the pathological condition of pulmonary hypertension, PLEKHH2 protein mutates or its expression level decreases, and Hic-5 captures more SMAD7, preventing SMAD7 from inhibiting SMAD2 / 3 activity, thereby enhancing the activity of the SAMD2 / 3 signaling pathway, promoting excessive proliferation of endothelial cells, accelerating the phenotype transition of endothelial cells to mesenchymal cells, and ultimately leading to pulmonary hypertension. Upregulating PLEKHH2 expression can prevent Hic-5 from binding to SMAD7, allowing more SMAD7 to be released into the cytoplasm, inhibiting SMAD2 / 3 phosphorylation, and thus treating pulmonary hypertension.

[0056] (3) Based on previous research results, this project further clarified the molecular mechanism by which PLEKHH2 protects pulmonary vascular remodeling and proposed and verified several new ideas for the treatment of pulmonary hypertension:

[0057] 1) The research results of the present invention show that Hic-5 is highly expressed in the case of pulmonary hypertension, capturing more SMAD7 protein and preventing it from inhibiting the function of SMAD2 / 3. Therefore, degrading Hic-5 is a potential strategy for treating pulmonary hypertension.

[0058] 2) Designing small molecule compounds that mimic the binding site of Hic-5 and PLEKHH2 to reduce the binding of Hic-5 to SMAD7 will be the direction of new drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is a genetic pedigree diagram showing that 6 of the 10 familial pulmonary hypertension families were found to carry known pathogenic gene mutations;

[0060] Figure 2 is a pedigree diagram showing that PLEKHH2 is a new potential pathogenic gene for pulmonary arterial hypertension, discovered through two families with hereditary pulmonary arterial hypertension. W / W: wild type, I1235T / W and S658F / W: both are PLEKHH2 heterozygous mutations;

[0061] Figure 3 shows the expression of the PLEKHH2 gene in various tissues from the NCBI database, wherein Figure A shows the expression of PLEKHH2 in various tissues of adults, and Figure B shows the expression of PLEKHH2 in various tissues during developmental stages;

[0062] FIG4 is a graph showing the expression levels of the PLEKHH2 gene in multiple organ tissues of rats detected by qPCR;

[0063] Figure 5 is an immunofluorescence image showing the main localization results of PLEKHH2, wherein blue is DAPI-stained nuclei, red is VWF, showing endothelial cells, and green is PLEKHH2 protein;

[0064] Figure 6 shows the results of Western blot detection of PLEKHH2 protein expression levels in pulmonary artery smooth muscle cells (PASMC), micropulmonary artery endothelial cells (PMEC), and pulmonary artery endothelial cells (PAEC), wherein, Figure A: Western blot results, Figure B: PLEKHH2 protein expression level statistics, *: P < 0.05, **: P < 0.01, n = 3;

[0065] Figure 7 shows the expression of PLEKHH2 gene in lung tissue of rats with pulmonary hypertension induced by monocrotaline, wherein, Figure A: statistical graph of mean pulmonary artery pressure (mPAP) of wild control rat group (WT group) and PAH rat model group (MCT group) constructed by subcutaneous injection of 60 mg / kg monocrotaline (MCT) in rats, Figure B: statistical graph of right ventricular hypertrophy index (RV / (LV+S)) of WT group and MCT group, Figure C: mRNA expression level of PLEKHH2 gene in WT group and MCT group, Figure D: Western blot results and statistical graph, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 vs WT group, n = 5;

[0066] Figure 8 shows the expression of PLEKHH2 gene in lung tissue of rats with pulmonary hypertension induced by hypoxia + Sugen stimulation, wherein, Figure A: Statistical graph of mean pulmonary artery pressure (mPAP) of wild-type control rat group (WT group) and PAH rat model group (Su+Hx group) constructed by subcutaneous injection of Su-5416 (20 mg / kg) for 3 weeks of hypoxia and 2 weeks of reoxygenation, Figure B: Statistical graph of right ventricular hypertrophy index (RV / (LV+S)) of WT group and Su+Hx group, Figure C: Result graph of mRNA expression level of PLEKHH2 gene in WT group and Su+Hx group, Figure D: Western blot result and statistical graph, **: P<0.01, ***: P<0.001, ****: P<0.0001 vs WT group, n=5;

[0067] FIG9 shows the results of immunofluorescence detection of PLEKHH2 gene expression in endothelial cells of lung tissues of patients with pulmonary hypertension, wherein Control: control lung tissue without pulmonary hypertension, PAH: lung tissue of patients with congenital heart disease and severe pulmonary hypertension;

[0068] Figure 10 shows the results of PLEKHH2 gene expression after siRNA-PLEKHH2 transfection of PAEC cells, wherein, Figure A: Western blot results, Figure B: PLEKHH2 protein expression level statistics, *: P < 0.05 vs si-NC group, n = 3;

[0069] Figure 11 is a graph showing the effect of knocking down the PLEKHH2 gene on the proliferation ability of PAEC cells detected by EDU cell proliferation experiment, wherein, Figure A: EDU staining results, Figure B: Statistical graph of EDU positive staining in the Nomo group, Figure C: Statistical graph of EDU positive staining in the Hypo group, Nomo group: normoxia (20% oxygen concentration), Hypo group: hypoxia (3% oxygen concentration), *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 vs si-NC group, n = 3;

[0070] Figure 12 shows the effect of knocking down PLEKHH2 on the expression of Bcl-2 and Bax in PAEC cells under normoxia by Western blot, wherein, Figure A: Western blot results (Bcl-2), Figure B: Bcl-2 expression level statistics, Figure C: Western blot results (Bax), Figure D: Bax expression level statistics, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 vs si-NC group, n = 3;

[0071] FIG13 is a diagram showing the results of a Matrigel angiogenesis assay detecting the effect of knocking down PLEKHH2 on the tube-forming ability of PAEC cells under normoxic or hypoxic conditions, wherein the Nomo group: normoxia (20% oxygen concentration) and the Hypo group: hypoxia (3% oxygen concentration);

[0072] Figure 14 shows the expression and phosphorylation changes of key proteins of the BMPR2 / TGF-β pathway in PAEC cells after knockdown of the PLEKHH2 gene by Western blot, wherein, Figure A: Western blot results, Figure B: PLEKHH2 expression level statistics, Figure C: BMPR2 expression level statistics, Figure D: p-SMAD1 / 5 / 8 / SMAD1 / 5 / 8, Figure E: SMAD1 / 5 / 8, Figure F: p-SMAD2 / 3 / SMAD2 / 3, Figure G: ID1, *: P < 0.05, **: P < 0.01 vs si-NC group, n = 1-3;

[0073] Figure 15 shows the results of in vivo elevation of the PLEKHH2 gene to effectively prevent pulmonary hypertension, wherein: Panel A: Schematic diagram of the experimental design; Panels B-C: Results of right cardiac catheterization testing of the effect of high PLEKHH2 expression on MCT-induced right ventricular systolic pressure (RVSP) in rats; Panels D-H: Results of ultrasound testing of the effect of high PLEKHH2 expression on MCT-induced right ventricular structure and function in rats, wherein: PAT / PET: pulmonary artery acceleration time (PAT) / pulmonary artery ejection time (PET), RVOT: right ventricular outflow tract width, TAPSE: tricuspid annular systolic excursion, RVID: right ventricular diameter, RVAW: right ventricular free wall thickness;

[0074] Figure 16 shows the results of in vivo elevation of the PLEKHH2 gene being effective in treating pulmonary hypertension, wherein: Panel A: Schematic diagram of the experimental design; Panels B-C: Results of right cardiac catheterization testing of the effect of PLEKHH2 treatment on MCT-induced right ventricular systolic pressure (RVSP) in rats; Panels D-H: Results of ultrasound testing of the effect of PLEKHH2 treatment on MCT-induced right ventricular structure and function in rats, wherein: PAT / PET: pulmonary artery acceleration time (PAT) / pulmonary artery ejection time (PET); RVOT: right ventricular outflow tract width; TAPSE: tricuspid annular systolic excursion; RVID: right ventricular diameter; RVAW: right ventricular free wall thickness;

[0075] Figure 17 is a Western blot analysis of the expression of PLEKHH2, Hic-5, and SMAD7 proteins in the lung tissues of MCT rats, wherein NC: control group subcutaneously injected with normal saline; MCT: model group subcutaneously injected with 60 mg / kg monocrotaline;

[0076] Figure 18 is immunofluorescence staining of pulmonary artery endothelial cells, wherein blue: cell nucleus; green: Hic-5; red: PLEKHH2;

[0077] Figure 19 is a schematic diagram of the protein structures of PLEKHH2, Hic-5 and SMAD7;

[0078] Figure 20 shows the predicted binding abilities and binding sites of Hic-5 to SMAD7 and Hic-5 to PLEKHH2 using Alpha-Fold-3 software, wherein Figure A: green represents the MH2 and N-terminal domains of the SMAD7 protein, and blue represents the full-length Hic-5 protein; Figure B: green represents the full-length Hic-5 protein, blue represents the FIRM domain of PLEKHH2, and purple represents the protein sequence that directly binds to Hic-5 and SMAD7;

[0079] Figure 21 shows the binding of PLEKHH2, SMAD7 and Hic-5 detected by Co-IP, wherein sh-P: PLEKHH2 gene is knocked down by sh-RNA; Ad-P: PLEKHH2 gene is overexpressed by adenovirus; Ad: empty adenovirus vector is used as control;

[0080] Figure 22 shows the binding of PLEKHH2, SMAD7 and Hic-5 in rat lung tissue after MCT stimulation detected by Co-IP, wherein WT: wild-type rats injected with normal saline as a control; WT+MCT: lung tissues of wild-type rats were obtained three weeks after injection of MCT (40 mg / kg); P-KO+MCT: lung tissues of PLEKHH2 gene knockout heterozygous rats were obtained three weeks after injection of MCT (40 mg / kg);

[0081] Figure 23 shows the binding of PLEKHH2, SMAD7, and Hic-5 in rat lung tissue after hypoxia stimulation detected by Co-IP, wherein WT: wild-type rats cultured in normoxia for three weeks as a control; WT+Hypo: lung tissues of wild-type rats obtained after three weeks of chronic hypoxia (10% oxygen concentration); P-KO+Hypo: lung tissues of PLEKHH2 gene knockout heterozygous rats obtained after three weeks of chronic hypoxia (10% oxygen concentration);

[0082] Figure 24 shows the binding of PLEKHH2, SMAD7, and Hic-5 in the lung tissues of rats with MCT-induced pulmonary hypertension after Co-IP detection of elevated PLEKHH2 expression in vivo, wherein WT: wild-type rats injected with normal saline; WT+MCT: wild-type rats injected with MCT (50 mg / kg) 4 weeks later; Ad-P+MCT: adenovirus prevention group, on the day of MCT injection, rats were first administered PLEKHH2 gene adenovirus via tracheal spray to upregulate PLEKHH2 gene expression, and PLEKHH2 gene adenovirus was administered for the second time 2 weeks later. The experiment was terminated at 4 weeks.

[0083] Figure 25 shows the binding of PLEKHH2, SMAD7, and Hic-5 to lung tissue in a rat model of pulmonary hypertension induced by hypoxia + Sugen after Co-IP detection of elevated PLEKHH2 expression in vivo, wherein WT: wild-type rats injected with normal saline and fed with normoxia for 5 weeks; WT+SuHx: disease model group, wild-type rats were subcutaneously injected with Su-5416 (20 mg / kg) and subjected to hypoxia (10% oxygen concentration) for 3 weeks and then reoxygenation for 2 weeks to establish a PAH rat model group; Ad-P+SuHx: prevention group, on the day of subcutaneous injection of Su-5416, rats were first administered with adenovirus encoding the PLEKHH2 gene via tracheal spray to upregulate PLEKHH2 gene expression, and adenovirus encoding the PLEKHH2 gene was administered a second time 3 weeks later, and the experiment was terminated at 5 weeks;

[0084] Figure 26 shows the activation of SMAD2 / 3 in the lung tissue of rats with MCT-induced pulmonary hypertension detected by Western blot after elevated PLEKHH2 expression in vivo, where WT: wild-type rats injected with normal saline; WT+MCT: wild-type rats 4 weeks after injection of MCT (50 mg / kg); Ad-P+MCT: adenovirus prevention group, on the day of MCT injection, the PLEKHH2 gene adenovirus was administered to the rats for the first time via tracheal spray to upregulate PLEKHH2 gene expression, and the PLEKHH2 gene adenovirus was administered for the second time 2 weeks later, and the experiment was terminated at 4 weeks. DETAILED DESCRIPTION

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In order to facilitate understanding of the present invention, the following terms involved in the present invention are explained here:

[0086] As used herein, the terms “include” or “comprising” mean including any one or more of the stated elements or components but not excluding other elements or components.

[0087] As used herein, the term "expression level" is the same as "level", and refers to the absolute or relative amount of the biomarker PLEKHH2 of the present invention. The expression level of the biomarker PLEKHH2 of the present invention can be determined by a variety of techniques. In particular, the absolute or relative amount of the biomarker PLEKHH2 of the present invention can be detected by methods well known to those skilled in the art.

[0088] As used herein, the term "primer" refers to a 7-50 nucleic acid sequence that can form a base pair complementary to a template strand and acts as a starting point for replicating the template strand. Primers are typically synthesized, but naturally occurring nucleic acids may also be used. The sequence of the primer does not necessarily need to be identical to that of the template, as long as it is sufficiently complementary and capable of hybridizing with the template. Additional features that do not change the basic properties of the primer may be incorporated. Examples of additional features that may be incorporated include, but are not limited to, methylation, capping, substitution of one or more nucleic acids by homologs, and modification between nucleic acids.

[0089] As used herein, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few to several hundred bases in length, that can specifically bind to mRNA and, through labeling, identify the presence of a specific mRNA. Probes can be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, and RNA probes.

[0090] As used herein, the term "antibody" refers to a specific immunoglobulin directed against an antigenic site. The antibody of the present invention refers to an antibody that specifically binds to the PLEKHH2 protein of the present invention, and the antibody can be produced according to conventional methods in the art. The form of the antibody includes polyclonal antibodies or monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2 and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising the antigen binding site of the antibody, and any other modified immunoglobulin molecules comprising the antigen binding site, as long as the antibody exhibits the desired biological binding activity.

[0091] As used herein, the term "peptide" refers to a class of substances that have the ability to highly bind to a target substance (the biomarker protein of the present invention) and do not denature during heat or chemical treatment. Moreover, due to its small size, it can be used as a fusion protein by attaching it to other proteins. Specifically, because it can be specifically attached to a polymer protein chain, it can be used as a diagnostic kit and drug delivery substance.

[0092] As used herein, the term "aptamer" refers to a polynucleotide composed of a specific type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and is capable of binding to a target molecule (the biomarker protein described herein) with high affinity and specificity. As mentioned above, aptamers can specifically bind to antigenic substances like antibodies, but are more stable and have a simpler structure than proteins, and are composed of easily synthesized polynucleotides. Therefore, they can be used in place of antibodies.

[0093] As used herein, the term "biomarker" is the same as "marker", and refers to an indicator of the phenotype of a patient (specifically a patient with pulmonary hypertension in the present invention), such as an indicator of a pathological state or possible responsiveness to a therapeutic agent, which can be detected in a biological sample of the patient. The biomarker includes but is not limited to: DNA, RNA, protein, small molecule metabolites, carbohydrates, glycolipid-based molecules, etc.; in a specific embodiment of the present invention, the biomarker is PLEKHH2.

[0094] As used herein, the term "treatment" generally refers to treatment of humans or animals (e.g., as used by veterinarians) to achieve some desired therapeutic effect, such as inhibiting the progression of a condition (including reducing the rate of progression or halting progression), ameliorating a condition, and curing a condition. Treatment as a preventative measure (e.g., prophylaxis) is also included. Use in patients who have not yet developed a condition but are at risk of developing it is also included in the term "treatment."

[0095] As used herein, the term "diagnosis" refers to the discovery, judgment or recognition of an individual's health status or condition based on one or more symptoms, data or other information related to the individual. The individual's health status can be diagnosed as healthy / normal (i.e., the absence of disease or illness), or can be diagnosed as unhealthy / abnormal (i.e., the presence of disease or illness). The terms diagnosis, early diagnosis, diagnosis and variations of these terms include early discovery of diseases / conditions associated with specific diseases or illnesses (specifically pulmonary hypertension in the present invention); characteristics or classification of the disease; discovery of the progression, cure or recurrence of the disease; discovery of the individual's response to the disease after treatment or treatment. In the present invention, the diagnosis and / or auxiliary diagnosis of pulmonary hypertension includes distinguishing between individuals who do not suffer from pulmonary hypertension and individuals who suffer from pulmonary hypertension.

[0096] As used herein, the term "pharmaceutical composition" may be in the form of any formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, lyophilized formulations, and suppositories. Furthermore, the pharmaceutical composition may be administered once or multiple times. In this case, the pharmaceutical composition may be administered in the form of a liquid formulation, powder, aerosol, capsule, or suppository.

[0097] The routes of administration of the pharmaceutical composition include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, rectal, etc. When administered orally, a coating can be formulated to protect the active ingredient in the pharmaceutical composition to prevent degradation in the stomach. In addition, the active ingredient can be administered by any device capable of being transferred to the target tissue. In a specific embodiment, the pharmaceutical composition provided by the present invention can be made into various dosage forms according to actual needs, and can be administered by a clinician based on factors such as the type, age, weight, and general condition of the subject, and the mode of administration to determine the dosage that is beneficial to the patient. The mode of administration can, for example, be injection or any other suitable mode of administration well known to those skilled in the art.

[0098] As used herein, the term "effective amount" refers to an amount that has a therapeutic effect or the amount required to produce a therapeutic effect in a subject. For example, a therapeutically or pharmaceutically effective amount of a drug refers to the amount of the drug required to produce the desired therapeutic effect, and the therapeutic effect can be reflected by the results of clinical trials, model animal studies, and / or in vitro studies. The pharmaceutically effective amount depends on several factors, including but not limited to: characteristic factors of the subject (such as height, weight, sex, age, and medication history), the severity of the disease, etc.

[0099] The present invention will be further described below in conjunction with specific examples, which are intended only to explain the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these examples without departing from the principles and aims of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods in the following examples, for which specific conditions are not specified, are generally tested under conventional conditions or under conditions recommended by the manufacturer.

[0100] Example 1 Collection and Study of Families with Hereditary Pulmonary Hypertension

[0101] 1. Experimental methods

[0102] From January 2021 to December 2022, a total of 10 hereditary pulmonary hypertension families were collected in the Genetics Clinic of Fuwai Hospital, Chinese Academy of Medical Sciences. Whole exome sequencing was performed on the probands of each of the above hereditary pulmonary hypertension families, and mutations were screened in 21 known pulmonary hypertension pathogenic genes (ACVRL1, BMPR2, ENG, GDF2, SMAD9, CAV1, ATP13A3, KCNK3, SOX17, EIF2AK4, TBX4, AQP1, SMAD4, SMAD1, KLF2, BMPR1B, KCNA5, ABCC8, KDR, TET2, GGCX).

[0103] The whole exome sequencing method is as follows: DNA was extracted from whole blood collected from participants using a DNA kit (Qiangen, Germany), and WES was performed according to the standards described in previous studies (see reference 1: Wang XJ, Lian TY, Jiang X, et al. Germline BMP9 mutation causes idiopathic pulmonary arterial hypertension. The European respiratory journal 2019; 53.; reference 2: Tan JS, Yan XX, Wu Y, et al. Rare variants in MTHFR predispose to occurrence and recurrence of pulmonary embolism. International journal of cardiology 2021; 331: 236-42.). DNA libraries were constructed using the SureSelect Human All Exon V6 kit and sequenced using the Illumina NovaSeq 6000 system. The average coverage depth of the target region for each sample was >100X, and more than 90% of the target bases were sequenced more than 20 times.

[0104] 2. Experimental results

[0105] The results showed that 6 families carried known pathogenic gene mutations of pulmonary arterial hypertension (see Figure 1), including 4 BMPR2 mutations, 1 TBX4 mutation and 1 ALK1 mutation (see Table 1), with a mutation detection rate of 60%.

[0106] Table 1 Pathogenic gene mutations found in families with hereditary pulmonary hypertension

[0107] NA: PolyPhen2 and SIFT cannot analyze variants in the in silico prediction programs for frameshift, nonsense, frameshift deletion, large deletion, and some genes with unknown causes. D: Deleterious in SIFT, possibly deleterious in PolyPhen2, P: Possibly deleterious.

[0108] Example 2 PLEKHH2 gene mutation is associated with familial pulmonary hypertension

[0109] 1. Experimental methods

[0110] For the four families in which no pathogenic gene mutations have been found, the present invention performed whole exome sequencing on all core members of families No. 7 and No. 8 to search for new potential pathogenic genes.

[0111] Family No. 7 has two patients with pulmonary hypertension. The proband is a 42-year-old woman. Right cardiac catheterization revealed a mean pulmonary artery pressure of 63 mmHg, a pulmonary vascular resistance of 12.1 Wood Units, and a negative acute pulmonary vasodilation test. The proband's second son (21 years old) also has pulmonary hypertension, with a mean pulmonary artery pressure of 87 mmHg, a pulmonary vascular resistance of 18.6 Wood Units, and a negative acute pulmonary vasodilation test. The proband's husband and eldest son are both healthy.

[0112] Family No. 8 also has two patients with pulmonary hypertension. The proband is a female patient diagnosed with pulmonary hypertension at age 38, with a mean pulmonary artery pressure of 43 mmHg, pulmonary vascular resistance of 5.3 Wood Units, and a negative acute pulmonary vasodilator test. The proband's daughter also has pulmonary hypertension, with a right ventricular systolic pressure of 60 mmHg assessed by ultrasound at age 14. The proband's husband is in good health.

[0113] In this example, whole-exome sequencing was performed on a family of four from Family 7 and a family of three from Family 8, and rare deleterious variants were selected using the following criteria:

[0114] (1) Co-segregation with the disease phenotype: the variant is shared by both patients but not by the control;

[0115] (2) the variant is located in the exon region or exon-intron splicing region of the gene;

[0116] (3) The mutation type is missense, stop gain, stop loss, or small indel, which should affect the amino acid coding;

[0117] (4) The variant frequency (MAF) in databases such as the 1000 Genomes and GnomAD East Asian populations is <0.1%;

[0118] (5) At least one of the three bioinformatics software programs, SIFT, PolyPhen-2, and Mutation taster, considers the mutation to be harmful.

[0119] 2. Experimental results

[0120] The results showed that after screening, a total of 61 genes were selected in Family 7 and 138 genes were selected in Family 8. The intersection of the rare variants in the two families revealed duplicated expression in only two genes: PLEKHH2 and PKDREJ. Since PKDREJ is not expressed in lung tissue, it was excluded, leaving only PLEKHH2 (see Figure 2).

[0121] Example 3 Verification of the genetic correlation between PLEKHH2 gene mutation and pulmonary hypertension

[0122] To clarify the correlation between PLEKHH2 gene mutations and the development of pulmonary arterial hypertension, this study enrolled 176 patients with idiopathic pulmonary arterial hypertension from the Fuwai Hospital of the Chinese Academy of Medical Sciences and performed whole-exome sequencing on all patients. Since all 176 patients were sporadic and had no family history of the disease as a reference, we adopted more stringent criteria when assessing the deleteriousness of genetic variants in the PLEKHH2 gene, as follows:

[0123] (1) The mutation is located in the exon region or exon-intron splicing region of the gene;

[0124] (2) The mutation type is missense, stop gain, stop loss, or small indel, which should affect the amino acid coding;

[0125] (3) The frequency of the variant should be extremely low, requiring that it is completely absent in the 1000 Genomes Chinese database and that its MAF in the GnomAD East Asian database is less than 1×10 - 5 ;

[0126] (4) If it is a missense mutation, all three bioinformatics software programs, SIFT, PolyPhen-2, and Mutation taster, must consider the mutation to be harmful.

[0127] Using the above criteria, we discovered 5 new patients carrying rare deleterious mutations in the PLEKHH2 gene, with a mutation rate of 2.8% (5 / 176 = 2.8%). The clinical phenotypes and PLEKHH2 gene mutations of the 5 patients are shown in Table 2. Specifically, the mutations include four missense mutations (c.1624C>T, c.1825G>A, c.2204A>G, c.3718A>T) and one truncating mutation (c.2389C>T). These mutations cause protein sequence changes to: p.P542S, p.A609T, p.Y735C, p.T1240S, and p.R797X (premature termination truncation at amino acid position 797). We verified the PLEKHH2 gene mutations carried by the 5 patients in the 1000 Genomes Chinese population and the GnomAD global population and found that the frequency of all mutations was less than 1×10 -5 These findings are consistent with the genetic characteristics of a rare pulmonary arterial hypertension (PAH) disease. All four missense mutations were consistently judged to be deleterious by three bioinformatics analysis software packages. We also analyzed these five patients for variants in 21 known pulmonary arterial hypertension-causing genes and found that they did not carry any known pulmonary arterial hypertension-causing mutations.

[0128] This example then analyzed the genetic burden of pulmonary hypertension (PAH) posed by rare variants in the PLEKHH2 gene. Using the same criteria used to screen for rare variants in patients with idiopathic PAH, a search was conducted in the GnomAD database for 76,156 controls for rare PLEKHH2 variants. 557 variants were identified, representing a mutation rate of only 0.07%. Rare PLEKHH2 variants were significantly more common in patients with PAH (P = 0.001, OR = 3.1). This preliminary study establishes that PLEKHH2 is a novel genetically associated gene for PAH, strongly implicated in the pathogenesis of PAH.

[0129] Table 2 Genotypes and clinical phenotypes of patients with familial and idiopathic pulmonary arterial hypertension carrying PLEKHH2 gene mutations

[0130] HPAH: hereditary pulmonary arterial hypertension; IPAH: idiopathic pulmonary arterial hypertension; mPAP: mean pulmonary artery pressure; PVR: pulmonary vascular resistance; CO: cardiac output. *: Patient 10 is a child; the 13.4 here is the normalized pulmonary vascular resistance (PVRi). Sex: F indicates female, M indicates male. MAF in 1000G_CHB: The frequency of this variant in the 1000 Genomes Chinese population (387 individuals). MAF in GnomAD: The frequency of this variant in the GnomAD database (V3.1.1, approximately 76,156 individuals).

[0131] Example 4 PLEKHH2 is specifically expressed in pulmonary artery endothelial cells

[0132] The NCBI database shows that the PLEKHH2 gene is highly expressed in lung tissue, both in the developmental stage and in the adult stage (see Figure 3A-B). In this example, 2-month-old wild-type SD rats were used for multiple organs (heart, lung, liver, spleen, kidney, brain, and skeletal muscle). In this example, GAPDH was used as an internal reference for relative quantification. The expression of PLEKHH2 in the various organs and tissues of the above rats was detected by qPCR. The results showed that the PLEKHH2 gene was highly expressed in lung tissue (see Figure 4). The specific primer sequences used in the qPCR experiment are as follows:

[0133] PLEKHH2 gene:

[0134] The forward primer is 5'-AGCGGACGACTCAAGACT-3' (SEQ ID NO: 1); the reverse primer is 5'-TGAACCGTTTGCTTGTTA-3' (SEQ ID NO: 2);

[0135] GAPDH gene:

[0136] The forward primer was 5'-CTCATGACCACAGTCCATGC-3' (SEQ ID NO: 3); the reverse primer was 5'-CACATTGGGGGTAGGAACAC-3' (SEQ ID NO: 4).

[0137] This example uses immunofluorescence to mark PLEKHH2 protein in human lung biopsy tissue and finds that it is mainly located in human pulmonary vascular endothelial cells, wherein blue is DAPI stained nucleus, red is VWF, showing endothelial cells, and green is PLEKHH2 protein (see Figure 5). In addition, this example cultured three types of human lung vascular structural cells in vitro, including pulmonary artery smooth muscle cells (PASMC), micropulmonary artery endothelial cells (PMEC), and pulmonary artery endothelial cells (PAEC). Western blot detected the expression level of PLEKHH2 protein in the above three cells. The results showed that PLEKHH2 protein was highly expressed in two endothelial cells and almost not expressed in smooth muscle cells (see Figure 6A-B). The characteristics of PLEKHH2 being highly expressed in lung tissue and mainly located in pulmonary vascular endothelial cells further suggest that it is closely related to pulmonary vascular pathophysiology.

[0138] Example 5 PLEKHH2 expression is significantly reduced under pulmonary hypertension pathological conditions

[0139] 1. Experimental methods

[0140] In order to clarify whether PLEKHH2 is involved in the pathological process of pulmonary hypertension, this example detected the expression of the PLEKHH2 gene in two animal models of pulmonary hypertension.

[0141] Firstly, a PAH rat model was established by subcutaneously injecting 60 mg / kg monocrotaline (MCT) into rats (MCT group). Three weeks later, the mean pulmonary artery pressure (mPAP) and right ventricular hypertrophy index (RV / (LV+S)) of wild-type control rats (WT group) and MCT group rats were detected. The expression levels of PLEKHH2 gene mRNA and PLEKHH2 protein in the lung tissues of wild-type control rats and MCT group rats were detected by qPCR and Western blot experiments, respectively.

[0142] Secondly, a PAH rat model (Su+Hx group) was established by subcutaneous injection of Su-5416 (20 mg / kg) in rats, followed by 3 weeks of hypoxia and 2 weeks of reoxygenation. Five weeks later, the mean pulmonary artery pressure (mPAP) and right ventricular hypertrophy index (RV / (LV+S)) of wild-type control group rats (WT group) and Su+Hx group rats were detected. The expression levels of PLEKHH2 gene mRNA and PLEKHH2 protein in the lung tissues of wild-type control group rats and Su+Hx group rats were detected by qPCR and Western blot experiments, respectively.

[0143] Finally, this example further collected lung tissues from patients with congenital heart disease and severe pulmonary hypertension and control lung tissues without pulmonary hypertension, and detected the expression of the PLEKHH2 gene in endothelial cells of the lung tissues of patients with pulmonary hypertension by immunofluorescence.

[0144] 2. Experimental results

[0145] The results showed that the expression of PLEKHH2 gene in the lung tissue of pulmonary hypertension rat models was significantly reduced in both monocrotaline-induced pulmonary hypertension rats and hypoxia + Sugen-induced pulmonary hypertension rats. Compared with wild-type control rats, the mean pulmonary artery pressure and right ventricular hypertrophy index of monocrotaline-induced pulmonary hypertension rats were significantly increased (see Figure 7A-B). The mRNA expression of PLEKHH2 gene in the MCT group decreased by 62% (see Figure 7C) and the protein expression decreased by 43% (see Figure 7D), which were extremely significant differences. Compared with wild-type control rats, the mean pulmonary artery pressure and right ventricular hypertrophy index of hypoxia + Sugen stimulation-induced pulmonary hypertension rats were significantly increased (see Figure 8A-B). The mRNA expression of PLEKHH2 gene in the hypoxia + Sugen group decreased by 76% (see Figure 8C) and the protein expression decreased by 50% (see Figure 8D), which were extremely significant differences. Furthermore, this example found that PLEKHH2 was also significantly decreased in lung tissue biopsies of patients with pulmonary hypertension (see FIG9 ), suggesting that this gene is closely related to the pathological remodeling of pulmonary hypertension.

[0146] Example 6 Knockdown of PLEKHH2 Gene Causes Mesenchymal Transition (EndoMT) Phenotype of Pulmonary Artery Endothelial Cells

[0147] 1. Experimental methods

[0148] Since the PLEKHH2 gene is significantly decreased under the pathological conditions of pulmonary hypertension, this example selected PAECs with the highest background expression of PLEKHH2 protein as model cells, and used siRNA to knock down PLEKHH2 protein to study the effect of PLEKHH2 on endothelial cell phenotype.

[0149] First, this example designs and synthesizes two siRNAs with different sequences for the target gene PLEKHH2, namely siRNA-1 and siRNA-2. Then, the siRNA is transfected into PAEC cells and the expression level of PLEKHH2 protein in the transfected PAEC cells is detected by Western blot. Secondly, this example uses an EDU cell proliferation assay to detect the effect of knocking down the PLEKHH2 gene on the proliferation ability of PAEC cells under normoxia (20% oxygen concentration) (Nomo) and hypoxia (3% oxygen concentration) (Hypo). Again, this example uses a Western blot assay to detect the effect of knocking down PLEKHH2 on the expression of Bcl-2 and Bax in PAEC cells under normoxia. Finally, this example uses a Matrigel angiogenesis assay to detect the effect of knocking down the PLEKHH2 gene on the tube formation ability of PAEC cells under normoxia (20% oxygen concentration) (Nomo) and hypoxia (3% oxygen concentration) (Hypo).

[0150] The sequence information of siRNA-1 and siRNA-2 are as follows:

[0151] siRNA-1:

[0152] The sense strand is 5'-GAGGAAAUGAGCAAGAUAUTT-3' (SEQ ID NO: 5); the antisense strand is 5'-AUAUCUUGCUCAUUUCCUCTT-3' (SEQ ID NO: 6);

[0153] siRNA-2:

[0154] The sense strand is 5'-GGCUUCUGAAAGUGAUUAUTT-3' (SEQ ID NO: 7); the antisense strand is 5'-AUAAUCACUUUCAGAAGCCTT-3' (SEQ ID NO: 8).

[0155] 2. Experimental results

[0156] The results showed that after siRNA-PLEKHH2 transfection of PAEC cells, the expression level of the PLEKHH2 gene was significantly reduced. The two interference fragments siRNA-1 and siRNA-2 reduced the expression of PLEKHH2 protein by 40%-50% (see Figure 10A-B). Whether under normoxia (20% oxygen concentration) or hypoxia (3% oxygen concentration) conditions, the proliferation ability of PAEC cells was significantly increased after knocking down the PLEKHH2 gene (see Figure 11A-C). Western blot analysis of cell proliferation and apoptosis-related pathway proteins showed that decreased PLEKHH2 gene expression led to a significant increase in the anti-apoptotic protein Bcl-2, while the expression of the pro-apoptotic protein Bax was reduced (see Figure 12A-D). The results of the endothelial cell tube formation experiment showed that, whether under normoxia or hypoxia, the tube formation ability of the siRNA-1 group that knocked down the PLEKHH2 gene was significantly reduced, and the siRNA-2 group was almost unable to form tubes (see Figure 13). In summary, knockdown of the PLEKHH2 gene can cause PAEC cells to overproliferate, resist apoptosis, and be difficult to form tubes, resulting in a significant endothelial-to-mesenchymal transition (EndoMT) phenotypic transition.

[0157] Example 7: Knockdown of the PLEKHH2 gene significantly reduces BMPR2 pathway activity in endothelial cells

[0158] 1. Experimental methods

[0159] To verify the relationship between PLEKHH2 and the BMPR2 / TGF-β pathway, this example used Western blot to detect changes in BMPR2 / TGF-β pathway activity after knocking down PLEKHH2 in endothelial cells (PAEC cells), including detection of changes in expression and phosphorylation of key proteins in the BMPR2 / TGF-β pathway (BMPR2, P-SMAD1 / 5 / 8, SMAD1 / 5 / 8, P-SMAD2 / 3, SMAD2 / 3, ID1). In this example, siRNA-2, which had a better effect on knocking down PLEKHH2 in Example 6, was selected to explore the molecular mechanism of action.

[0160] 2. Experimental results

[0161] The results showed that decreased PLEKHH2 expression led to downregulation of BMPR2 expression in endothelial cells, decreased SMAD1 / 5 / 8 phosphorylation, and decreased ID1 expression. On the other hand, SMAD2 / 3 phosphorylation of the TGF-β pathway increased significantly (see Figure 14A-G). Therefore, PLEKHH2 is likely to be a key gene regulating the balance of the BMPR2 / TGF-β signaling pathway in endothelial cells. Knocking down PLEKHH2 will lead to weakened BMPR2 signaling and enhanced TGF-β signaling, thereby leading to vascular remodeling in pulmonary hypertension.

[0162] Example 8: Elevating the PLEKHH2 gene in vivo can effectively prevent pulmonary hypertension

[0163] 1. Experimental methods

[0164] PLEKHH2 mutation or reduced expression can promote the occurrence and development of pulmonary hypertension, suggesting that PLEKHH2 itself should be a "good gene" that protects pulmonary blood vessels. This example uses an in vivo animal model to further study whether increasing PLEKHH2 can prevent pulmonary hypertension.

[0165] A pulmonary hypertension rat model was constructed by intraperitoneal injection of monocrotaline (MCT, 50 mg / kg) in 8-week-old rats. On the day of MCT injection, the PLEKHH2 gene adenovirus (adenovirus-PLEKHH2) was administered to the rats for the first time by tracheal spray to upregulate PLEKHH2 gene expression. At 10 weeks of age, the PLEKHH2 gene adenovirus (adenovirus-PLEKHH2) was administered for the second time. The experiment was terminated at 12 weeks of age to detect the preventive effect of upregulating PLEKHH2 gene expression on MCT-induced pulmonary hypertension rats. The specific experimental design schematic is shown in Figure 15A, wherein the control group (control) was adenovirus-vehicle and the experimental group (case) was adenovirus-PLEKHH2. It is worth noting that the activity of adenovirus can only be maintained for two weeks in rats, so two adenovirus injections were required within the four-week period of this experiment to observe the preventive effect.

[0166] 2. Experimental results

[0167] The results showed that preventive administration of PLEKHH2 significantly decreased the right ventricular pressure in rats with MCT-induced pulmonary hypertension (see Figure 15B-C), significantly reduced the right ventricular wall thickness, right ventricular diameter, and right ventricular outflow tract width, and significantly increased the systolic displacement of the tricuspid annulus (see Figure 15D-H), indicating that high expression of PLEKHH2 can effectively prevent MCT-induced pulmonary hypertension.

[0168] Example 9: Elevating the PLEKHH2 gene in vivo can effectively treat pulmonary hypertension

[0169] 1. Experimental methods

[0170] After confirming that high expression of PLEKHH2 has the function of preventing pulmonary hypertension, this example further tested whether PLEKHH2 can treat pulmonary hypertension.

[0171] A pulmonary hypertension rat model was established using 8-week-old rats by intraperitoneal injection of monocrotaline (MCT, 50 mg / kg). The pulmonary hypertension of the rats had increased in the second week after MCT injection. PLEKHH2 gene adenovirus (adenovirus-PLEKHH2) was administered into the rats by tracheal spray. PLEKHH2 gene adenovirus was administered again in the third week. The experiment was terminated in the fourth week to detect the therapeutic effect of upregulating PLEKHH2 gene expression on MCT-induced pulmonary hypertension rats. The specific experimental design is shown in Figure 16A, wherein the control group (control) is adenovirus-vehicle and the experimental group (case) is adenovirus-PLEKHH2.

[0172] 2. Experimental results

[0173] The results showed that therapeutic administration of PLEKHH2 significantly reduced the pulmonary hypertension that had already occurred in rats with MCT-induced pulmonary hypertension (see 16B-C), significantly reduced the right ventricular wall thickness, right ventricular diameter, and right ventricular outflow tract width, and significantly increased the systolic displacement of the tricuspid annulus (see Figure 16D-F), indicating that high expression of PLEKHH2 can effectively treat MCT-induced pulmonary hypertension.

[0174] Example 10 In a rat model of pulmonary hypertension, the expression of PLEKHH2 and SMAD7 is reduced, while the expression of Hic-5 is increased

[0175] The roles of Hic-5 and SMAD7 in pulmonary hypertension are currently completely unknown. To clarify whether these two proteins are involved in the pathological process of pulmonary hypertension, this example used conventional methods to construct a classic monocrotaline (MCT)-induced pulmonary hypertension animal model and detected the expression of PLEKHH2, Hic-5, and SMAD7 proteins.

[0176] We established a PAH rat model (MCT group) by subcutaneously injecting 60 mg / kg monocrotaline (MCT) into rats, and subcutaneously injected normal saline into the control group. Three weeks later, lung tissues from the control and MCT groups were collected for Western blot analysis (Figure 17). The results showed that in the MCT model group, PLEKHH2 protein was significantly reduced (this result is completely consistent with the results in the previous example), Hic-5 expression was increased, and SMAD7 expression was decreased, suggesting that these three proteins are involved in vascular remodeling in pulmonary arterial hypertension.

[0177] Example 11 Co-localization of PLEKHH2 and Hic-5 in Pulmonary Vascular Endothelial Cells

[0178] Previous literature reported that PLEKHH2 and Hic-5 co-localize in podocytes (i.e., epithelial cells in the visceral layer of the renal capsule), but whether they co-localize in pulmonary vascular endothelial cells is completely unknown.

[0179] To this end, we cultured pulmonary artery endothelial cells (PAEC) in vitro and confirmed through immunofluorescence staining that both Hic-5 and PLEKHH2 were highly expressed in PAEC, with their subcellular localization mainly concentrated in the cytoplasm, and their localization was highly overlapping (Figure 18).

[0180] Example 12 Bioinformatics prediction of PLEKHH2 and SMAD7 competitive binding to the LIM3 domain of Hic-5

[0181] Hic-5 protein has seven major functional domains, namely three LD domains and four LIM domains; SMAD7 has two major domains: the N-terminal domain and the MH2 domain; PLEKHH2 has five domains: the CC domain, two PH domains, a MyTH domain and a FIRM domain (Figure 19).

[0182] Literature reports that the MH2 and N-terminal domains of the SMAD7 protein directly bind to the LIM3 domain (328-380aa) of the Hic-5 protein (Oncogene. 2008 November 20; 27(54): 6791–6805). We verified the binding ability of the above two protein domains using Alpha-Fold-3 software (Figure 20A).

[0183] According to the literature, the FIRM domain of PLEKHH2 can bind to Hic-5 (Kidney International (2012) 82, 1071–1083), but it is not clear which domain of Hic-5 it binds to.

[0184] To this end, we entered the FIRM domain of PLEKHH2 and the full-length protein sequence of Hic-5 into the Alpha-Fold-3 software and found that the FIRM domain of PLEKHH2 also directly binds to the LIM3 domain of Hic-5. Further, we marked the protein sequence that binds Hic-5 and SMAD7 with purple, and this position is located in the binding area between the FIRM domain of PLEKHH2 and the LIM3 domain of Hic-5 (Figure 20B). This suggests that PLEKHH2 and SMAD7 are likely to bind to the LIM3 region of Hic-5, and there is a competitive binding relationship between the two.

[0185] Example 13: PLEKHH2 and SMAD7 Competitively Bind to Hic-5 at the Cellular Level

[0186] To determine whether PLEKHH2 and SMAD7 competitively bind to Hic-5, we cultured pulmonary vascular endothelial cells, knocked down the PLEKHH2 gene using sh-RNA (sh-P), and overexpressed the PLEKHH2 gene using adenovirus (Ad-P). We then used Co-IP experiments to determine the changes in the binding of Hic-5 and SMAD7 after changes in PLEKHH2 expression.

[0187] As shown in Figure 21, sh-RNA indeed knocked down PLEKHH2 gene expression in pulmonary vascular endothelial cells and reduced the binding of PLEKHH2 protein to Hic-5. At this time, the expression level of SMAD7 did not change significantly, but the number of binding to Hic-5 increased significantly. In contrast, after adenovirus overexpressed the PLEKHH2 gene, the number of binding to Hic-5 increased significantly, and the binding of SMAD7 to Hic-5 decreased significantly.

[0188] The above cell experiments clearly show that the binding of PLEKHH2, SMAD7 and Hic-5 proteins is significantly negatively correlated, and there is an obvious competitive relationship between the two.

[0189] Example 14: PLEKHH2 and SMAD7 Competitively Bind to Hic-5 at the Animal Level in Vivo

[0190] To further clarify whether PLEKHH2 and SMAD7 competitively bind to Hic-5 in animals, we established pulmonary hypertension rat models using subcutaneous injection of monocrotaline and chronic hypoxia, respectively. After successful modeling, lung tissue was obtained from the rats and the binding of PLEKHH2 and SMAD7 to Hic-5 was detected by Co-IP experiments.

[0191] (1) MCT treatment group

[0192] We performed three groups of rats: 1) wild-type rats injected with saline as a control (WT), 2) wild-type rats injected with MCT (40 mg / kg) for three weeks (WT+MCT), and 3) PLEKHH2 knockout heterozygous rats injected with MCT (40 mg / kg) for three weeks (P-KO+MCT). Total lung tissue protein was collected and assayed for PLEKHH2 and SMAD7 protein levels captured by the Hic-5 antibody.

[0193] Consistent with the previous study results of the present application, PLEKHH2 protein significantly reduces in the lung tissue of WT+MCT group rats, and reduces more in P-KO+MCT group rats. Accordingly, the PLEKHH2 protein combined with Hic-5 is the highest in WT, significantly reduces in WT+MCT, and reduces more in P-KO+MCT. In contrast, the combination of SMAD7 protein and Hic-5 significantly increases in WT+MCT compared to WT, and increases more in P-KO+MCT (Figure 22).

[0194] (2) Chronic hypoxia group

[0195] We established three groups of rats: 1) wild-type rats (WT) housed in normoxia, 2) wild-type rats subjected to chronic hypoxia (10% oxygen concentration) for three weeks (WT+Hypo), and 3) PLEKHH2 knockout heterozygous rats subjected to chronic hypoxia (10% oxygen concentration) for three weeks (P-KO+Hypo). Total lung tissue protein was collected and assayed for PLEKHH2 and SMAD7 protein levels captured by the Hic-5 antibody.

[0196] Consistent with the results of the MCT group, PLEKHH2 protein was significantly reduced in the lung tissue of rats in the WT+Hypo group and was more reduced in rats in the P-KO+Hypo group. Correspondingly, the PLEKHH2 protein bound to Hic-5 was highest in WT, significantly reduced in WT+Hypo, and more reduced in P-KO+Hypo. In contrast, the binding of SMAD7 protein to Hic-5 increased in WT+Hypo compared to WT, and increased extremely significantly in P-KO+Hypo (Figure 23).

[0197] Therefore, using two animal models of pulmonary hypertension, we confirmed that the binding of PLEKHH2 and SMAD7 proteins to Hic-5 in rat lung tissue is significantly negatively correlated, indicating a clear competitive relationship between the two proteins. Reduced PLEKHH2 expression promotes the binding of Hic-5 to SMAD7, reducing free SMAD7 protein. This molecular mechanism is consistent with our previous findings that PLEKHH2 knockout exacerbates the phenotype of animal models of pulmonary hypertension, suggesting that an imbalance between PLEKHH2, Hic-5, and SMAD7 is a key mechanism in the development and progression of pulmonary hypertension.

[0198] Example 15: Elevated PLEKHH2 expression reduces Hic-5 binding to SMAD7 in the setting of pulmonary hypertension

[0199] Previous work has shown that reduced PLEKHH2 protein expression promotes increased binding of Hic-5 to SMAD7. So, can supplementing PLEKHH2 inhibit Hic-5 and SMAD7 binding in vivo? We established two pulmonary hypertension rat models using MCT and Sugen combined with hypoxia, respectively. We elevated gene expression in lung tissue by tracheal aerosolization of PLEKHH2 adenovirus. Three weeks later, lung tissue was collected from the rats and Co-IP assays were performed to examine the binding of PLEKHH2 and SMAD7 to Hic-5.

[0200] (1) Rats in the MCT group

[0201] We collected lung tissues from three groups of rats: 1) wild-type rats (WT) injected with normal saline, 2) wild-type rats injected with MCT (50 mg / kg) 4 weeks later (WT+MCT); 3) rats that were given adenovirus with the PLEKHH2 gene for the first time via tracheal spray on the day of MCT injection to upregulate PLEKHH2 gene expression, and adenovirus with the PLEKHH2 gene for the second time 2 weeks later, and the experiment was terminated at 4 weeks later (Ad-P+MCT).

[0202] As shown in Figure 24, the PLEKHH2 protein in the WT+MCT disease group rats was significantly reduced, and the binding of Hic-5 to SMAD7 was significantly enhanced. When the PLEKHH2 protein was significantly increased by adenovirus, the binding level of Hic-5 to SMAD7 was reduced to the normal control level.

[0203] (2) Rats in the Sugen+hypoxia group

[0204] We collected lung tissue from three groups of rats: 1) wild-type rats injected with normal saline and fed with normoxia for 5 weeks (WT); 2) wild-type rats subcutaneously injected with Su-5416 (20 mg / kg) and subjected to hypoxia (10% oxygen concentration) for 3 weeks followed by reoxygenation for 2 weeks to create a PAH rat model group (WT+SuHx); 3) on the day of subcutaneous injection of Su-5416, the rats were first administered with adenovirus encoding the PLEKHH2 gene via tracheal spray to upregulate PLEKHH2 gene expression, and the PLEKHH2 gene adenovirus was administered a second time at 3 weeks, and the experiment was terminated at 5 weeks (Ad-P+SuHx).

[0205] As shown in Figure 25, PLEKHH2 protein was significantly reduced in rats in the WT+SuHx disease group, and the binding of Hic-5 to SMAD7 was significantly enhanced. When PLEKHH2 protein was significantly increased by adenovirus, the binding level of Hic-5 to SMAD7 was reduced back to near the normal control level.

[0206] Therefore, we confirmed in two animal models of pulmonary hypertension that exogenous supplementation of PLEKHH2 protein significantly reduced Hic-5 binding to SMAD7 and increased free SMAD7 protein in the setting of pulmonary hypertension. This molecular mechanism is consistent with our previous research findings that PLEKHH2 gene supplementation can prevent or treat the phenotypes of pulmonary hypertension animal models, suggesting that restoring the balance between PLEKHH2, Hic-5, and SMAD7 is an important strategy for treating or preventing pulmonary hypertension.

[0207] Example 16 PLEKHH2 prevents pulmonary hypertension by inhibiting SMAD2 / 3 hyperphosphorylation

[0208] As mentioned above, SMAD7 is an important SMAD2 / 3 activity inhibitor. To confirm whether SMAD7 protein is released after PLEKHH2 competitively binds to Hic-5 and is active, we use the MCT group rat lung tissue in the previous embodiment to detect PLEKHH2 protein expression and SMAD2 / 3 phosphorylation. As shown in Figure 26, after MCT stimulation, PLEKHH2 significantly reduces, and the total protein of SMAD2 / 3 significantly reduces, and phosphorylated SMAD2 / 3 significantly increases. After using adenovirus to highly express PLEKHH2, the total protein of SMAD2 / 3 recovers and is close to the wild-type rat level, and the degree of phosphorylation significantly reduces.

[0209] We have discovered for the first time that PLEKHH2 and SMAD7 competitively bind to the Hic-5 protein in lung tissue. In the pathology of pulmonary hypertension, PLEKHH2 protein mutations or reduced expression allow Hic-5 to capture more SMAD7, preventing SMAD7 from inhibiting SMAD2 / 3 activity. This in turn enhances SAMD2 / 3 signaling, promotes excessive endothelial cell proliferation, accelerates the endothelial cell mesenchymal phenotype transition, and ultimately leads to pulmonary hypertension. Upregulating PLEKHH2 expression can prevent Hic-5 from binding to SMAD7, allowing more SMAD7 to be released into the cytoplasm, inhibiting SMAD2 / 3 phosphorylation, and thus treating pulmonary hypertension.

[0210] Based on previous research results, this project further clarified the molecular mechanism by which PLEKHH2 protects pulmonary vascular remodeling and proposed and verified several new ideas for the treatment of pulmonary arterial hypertension:

[0211] (1) Our results showed that Hic-5 is highly expressed in pulmonary hypertension, capturing more SMAD7 protein and preventing it from inhibiting the function of SMAD2 / 3. Therefore, degradation of Hic-5 is a potential strategy for the treatment of pulmonary hypertension.

[0212] (2) Designing small molecule compounds that mimic the binding site of Hic-5 and PLEKHH2 to reduce the binding of Hic-5 and SMAD7 will be the direction of new drug research and development.

[0213] In addition, the present invention has confirmed through experiments that the above-mentioned small molecule compound that inhibits the binding of Hic-5 and SMAD7 can be used for the effective treatment of pulmonary arterial hypertension.

[0214] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. Use of an agent that inhibits the binding of Hic-5 to SMAD7 and / or an agent that inhibits Hic-5 in the preparation of a drug for preventing, treating, alleviating and / or improving pulmonary hypertension.

2. The use according to claim 1, characterized in that: The reagents include small molecule compounds, polypeptides, peptide mimetics, nucleic acid molecules, proteins, antibodies, antibody mimetics, fusion proteins, protein analogs, gene delivery vectors, protein delivery vectors, aptamers, shRNA, siRNA, miRNA, antisense nucleic acids, CRISPR gene editing reagents and / or any combination thereof targeting the Hic-5 and SMAD7 binding sites and / or targeting Hic-5.

3. The use according to claim 1, characterized in that: The Hic-5 and SMAD7 binding site is the LIM3 domain of Hic-5.

4. The use according to claim 1, characterized in that: The agent is capable of competing with SMAD7 for binding to the LIM3 domain of Hic-5.

5. The use according to claim 1, characterized in that: The reagents include PLEKHH2 and / or a PLEKHH2 promoter.

6. The use according to claim 5, characterized in that: The PLEKHH2 and / or PLEKHH2 promoter include PLEKHH2 gene, PLEKHH2 mRNA, PLEKHH2 cDNA, PLEKHH2 protein, peptide segment of PLEKHH2 protein, analog of PLEKHH2 protein, active fragment of any of the foregoing, vector expressing PLEKHH2, nanoparticles carrying PLEKHH2 gene, viral vector carrying PLEKHH2 gene, PEG-modified protein encapsulating PLEKHH2 gene or protein, protein microspheres encapsulating PLEKHH2 gene or protein, liposomes encapsulating PLEKHH2 gene or protein, extracellular vesicles encapsulating PLEKHH2 gene or protein and / or any combination thereof.

7. The use according to claim 5, characterized in that: The PLEKHH2 and / or PLEKHH2 promoter prevents, treats, alleviates and / or improves pulmonary arterial hypertension by inhibiting SMAD2 / 3 hyperphosphorylation.

8. Use of an agent that inhibits the binding of Hic-5 to SMAD7 and / or an agent that inhibits Hic-5 in preventing, treating, alleviating and / or improving pulmonary arterial hypertension.

9. The use according to claim 8, characterized in that: The reagent is a reagent for inhibiting the binding of Hic-5 to SMAD7 and / or a reagent for inhibiting Hic-5 as described in any one of claims 1 to 7.

10. Use of PLEKHH2 and / or a PLEKHH2 promoter in the preparation of a medicament for preventing, treating, alleviating and / or improving pulmonary hypertension.

11. The use according to claim 10, characterized in that: The PLEKHH2 includes PLEKHH2 gene, PLEKHH2 mRNA, PLEKHH2 cDNA, PLEKHH2 protein, active fragments of any of the foregoing and / or any combination thereof.

12. The use according to claim 10, characterized in that: The PLEKHH2 promoter includes a substance that increases the level of PLEKHH2, a substance that enhances the activity of PLEKHH2, a substance that delays the metabolism of PLEKHH2 and / or any combination thereof.

13. The use according to claim 12, characterized in that: The PLEKHH2 promoter includes naturally purified substances, modified naturally purified substances, semi-synthetic substances and / or chemically synthesized substances.

14. The use according to claim 13, characterized in that The PLEKHH2 promoter includes PLEKHH2 protein, peptide fragments of PLEKHH2 protein, analogs of PLEKHH2 protein, vectors expressing PLEKHH2, nanoparticles carrying PLEKHH2 gene, viral vectors carrying PLEKHH2 gene, PEG-modified proteins encapsulating PLEKHH2 gene or protein, protein microspheres encapsulating PLEKHH2 gene or protein, liposomes encapsulating PLEKHH2 gene or protein, extracellular vesicles encapsulating PLEKHH2 gene or protein and / or any combination thereof.

15. The use according to claim 10, characterized in that The PLEKHH2 and / or PLEKHH2 promoter can reduce right ventricular systolic pressure, alleviate right ventricular hypertrophy, increase tricuspid valve ring systolic displacement, and alleviate pulmonary artery vascular remodeling.

16. Use of PLEKHH2 and / or PLEKHH2 promoter in preventing, treating, alleviating and / or improving pulmonary arterial hypertension.

17. The use according to claim 16, characterized in that The PLEKHH2 and / or PLEKHH2 promoter is the PLEKHH2 and / or PLEKHH2 promoter described in any one of claims 10-15.

18. A pharmaceutical composition for preventing, treating, alleviating and / or improving pulmonary hypertension, characterized in that: The pharmaceutical composition comprises the agent for inhibiting the binding of Hic-5 to SMAD7 and / or the agent for inhibiting Hic-5 as described in any one of claims 1 to 7, and / or the PLEKHH2 and / or PLEKHH2 promoter as described in any one of claims 10 to 15.

19. The pharmaceutical composition according to claim 18, characterized in that The pharmaceutical composition may further comprise other drugs for preventing, treating, alleviating and / or improving pulmonary hypertension.

20. The pharmaceutical composition according to claim 19, characterized in that The drugs include calcium channel blockers, prostacyclin drugs, endothelin receptor antagonists, phosphodiesterase-5 inhibitors and / or guanylate cyclase agonists.

21. The pharmaceutical composition according to claim 18, characterized in that The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipient.

22. A pharmaceutical preparation for preventing, treating, alleviating and / or improving pulmonary hypertension, characterized in that: The pharmaceutical preparation comprises the pharmaceutical composition according to any one of claims 18 to 21.

23. The pharmaceutical preparation according to claim 22, characterized in that The dosage forms of the pharmaceutical preparation include injection, lyophilized powder, solution, tablet, capsule, granule, ointment, cream, gel, suspension, oral solution, controlled release dosage form, nanocrystal preparation, microemulsion and / or solid dispersion.

24. Use of the LIM3 domain of Hic-5 in screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary arterial hypertension.

25. Use of PLEKHH2 and / or Hic-5 in screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary arterial hypertension.

26. Use of Hic-5 and / or SMAD7 in screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary arterial hypertension.

27. Use of SMAD7 in the preparation of a drug for preventing, treating, alleviating and / or improving pulmonary arterial hypertension.

28. Use of a reagent for detecting the expression level of PLEKHH2 in a sample in the preparation of a product for diagnosing and / or assisting in the diagnosis of pulmonary arterial hypertension.

29. The use according to claim 28, characterized in that The reagents include a reagent for detecting the expression level of PLEKHH2 mRNA in a sample and a reagent for detecting the expression level of PLEKHH2 protein in a sample.

30. The use according to claim 29, characterized in that The reagent for detecting the expression level of PLEKHH2 mRNA in a sample includes primers for specifically amplifying PLEKHH2 and / or probes for specifically recognizing PLEKHH2.

31. The use according to claim 29, characterized in that The reagent for detecting the expression level of PLEKHH2 protein in a sample includes a binding agent that specifically binds to the protein encoded by PLEKHH2.

32. The use according to claim 30, characterized in that The binding agent includes an antibody, an antibody functional fragment, an agglutinating agent, a receptor and / or a coupled antibody that specifically binds to a protein encoded by PLEKHH2.

33. A method for screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary hypertension, characterized in that: The method comprises the following steps: (a) treating a system expressing or containing the PLEKHH2 gene with a test substance; (b) detecting the expression of the PLEKHH2 gene in the system; (c) Selecting test substances that can increase the expression level of the PLEKHH2 gene as candidate drugs.

34. The method according to claim 33, characterized in that The system includes a cell system, a subcellular system, a solution system, a tissue system, an organ system and / or an animal system.

35. The method according to claim 33, characterized in that The substances to be tested include substances that increase the level of PLEKHH2, substances that enhance the activity of PLEKHH2, substances that delay the metabolism of PLEKHH2 and / or any combination thereof.

36. A method for screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary hypertension based on the LIM3 domain of Hic-5, characterized in that: The method comprises the following steps: screening an agent capable of binding to the LIM3 domain of Hic-5 as a candidate drug.

37. The method according to claim 36, characterized in that The reagents include small molecule compounds, polypeptides, peptide mimetics, nucleic acid molecules, proteins, antibodies, antibody mimetics, fusion proteins, protein analogs, gene delivery vectors, protein delivery vectors, aptamers, shRNA, siRNA, miRNA, antisense nucleic acids, CRISPR gene editing reagents and / or any combination thereof targeting the Hic-5 and SMAD7 binding sites and / or targeting Hic-5.

38. A method for screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary hypertension based on PLEKHH2 and / or Hic-5, characterized in that: The method comprises the following steps: simulating the binding site of Hic-5 and PLEKHH2, and designing and / or screening agents capable of binding to the binding site as candidate drugs.

39. The method according to claim 38, characterized in that The reagents include small molecule compounds, polypeptides, peptide mimetics, nucleic acid molecules, proteins, antibodies, antibody mimetics, fusion proteins, protein analogs, gene delivery vectors, protein delivery vectors, aptamers, shRNA, siRNA, miRNA, antisense nucleic acids, CRISPR gene editing reagents and / or any combination thereof targeting the Hic-5 and SMAD7 binding sites and / or targeting Hic-5.

40. A method for screening candidate drugs for preventing, treating, alleviating and / or improving pulmonary hypertension based on Hic-5 and / or SMAD7, characterized in that, The method comprises the following steps: simulating the binding site of Hic-5 and SMAD7, and designing and / or screening agents that can bind to the binding site as candidate drugs.

41. The method according to claim 40, characterized in that The reagents include small molecule compounds, polypeptides, peptide mimetics, nucleic acid molecules, proteins, antibodies, antibody mimetics, fusion proteins, protein analogs, gene delivery vectors, protein delivery vectors, aptamers, shRNA, siRNA, miRNA, antisense nucleic acids, CRISPR gene editing reagents and / or any combination thereof targeting the Hic-5 and SMAD7 binding sites and / or targeting Hic-5.

42. A product for diagnosing and / or assisting in the diagnosis of pulmonary hypertension, characterized in that: The product comprises the reagent described in any one of claims 28-32.

43. The product according to claim 42, characterized in that The product also includes reagents for detecting the expression level of PLEKHH2 in a sample by sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology and / or protein immunoassay technology.

44. The product according to claim 42, characterized in that The products include test kits, chips and / or test strips.

45. A system / device for diagnosing and / or assisting diagnosis of pulmonary hypertension, characterized in that: The system / device includes a processor, an input module, and an output module; Among them, the processor is used to perform logical operations on the input information using bioinformatics methods; the input module is used to input the expression level of PLEKHH2 in the subject's sample, and contains a computer-readable medium of instructions, which, when executed by the processor, executes an algorithm on the input expression level of PLEKHH2; the output module is used to output whether the subject suffers from pulmonary arterial hypertension or the risk of suffering from pulmonary arterial hypertension.

46. ​​A method for preventing, treating, alleviating and / or improving pulmonary hypertension, characterized in that: The method comprises the following steps: administering to a subject in need thereof an effective amount of an agent that inhibits the binding of Hic-5 to SMAD7 and / or an agent that inhibits Hic-5 as described in any one of claims 1-7, PLEKHH2 and / or a PLEKHH2 promoter as described in any one of claims 10-15, a pharmaceutical composition as described in any one of claims 18-21, and / or a pharmaceutical preparation as described in claim 22 or 23.

47. A method for diagnosing and / or assisting in the diagnosis of pulmonary hypertension, characterized in that: The method comprises the following steps: detecting the expression level of PLEKHH2 in a sample derived from a subject; if the expression level of PLEKHH2 in the sample derived from the subject is significantly lower than that of a normal person, the subject is diagnosed as a patient with pulmonary hypertension.

48. The method according to claim 47, characterized in that The sample includes blood, tissue, blood-derived cells, serum, plasma, lymph fluid, synovial fluid, cells and / or any combination thereof derived from a subject.

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